Project Summary ? Kim Lentiviruses including HIV-1, HIV-2 and SIV replicate in both activated/dividing CD4+ T cells and terminally differentiated/non-dividing myeloid cells such as macrophages and microglia. While HIV-1 rapidly replicates in activated CD4+ T cells, HIV-1 replication in macrophages is kinetically suppressed. In 2004, our HIV-1 reverse transcriptase (RT) based dNTP assay revealed that macrophages harbor an extremely low dNTP substrate concentration (20-40 nM), which kinetically restricts viral reverse transcription. In 2012, we reported that a host lentivirus restriction factor, SAMHD1, which hydrolyzes dNTPs, is responsible for the limited dNTP pools observed in macrophages. However, unlike HIV-1, HIV-2 (and SIVsm) efficiently replicates even in macrophages due to its accessary protein, Vpx, which counteracts SAMHD1, elevates the dNTP concentration, and then accelerates the proviral DNA synthesis kinetics in the non-dividing target cell types. We previously reported that RTs of retroviruses are kinetically adapted to the availability of cellular dNTPs in their target cell types. This idea was further supported by our recent studies that RT of HIV-1 exhibits higher DNA synthesis efficiency at the low macrophage dNTP concentrations, compared to RTs of HIV-2 and SIV that replicate at high dNTP concentrations even in macrophages. Indeed, our initial pre-steady state kinetic study revealed that HIV-1 RTs have similar Kd values (dNTP binding affinity), but significantly faster kpol step, which consists of conformational change, Kconf, and chemistry step, Kchem, compared to HIV-2/SIV RTs. In this proposal, first, we will test our new hypothesis that HIV-1 RT evolved to have faster Kconf step than HIV- 2/SIV RTs in order to effectively support proviral DNA synthesis at the lower dNTP concentrations in the non- dividing cells. Second, we reported that HIV-1 RT is highly error-prone due to its unique high mismatch extension capability. Here, we hypothesize HIV-1 can form stable complex with mismatch primer, which will enable us to solve the structure of HIV-1 RT complex with mismatch primer that can structurally elucidate the low fidelity nature of HIV-1 RT. Third, we previously reported that, due to the greater concentration discrepancy between dNTPs and rNTPs (~mM) in macrophages, HIV-1 RT frequently incorporates the non- canonical/mutagenic rNTPs in macrophages. We hypothesize that HIV-2/SIVs, which replicate at high dNTP concentrations even in macrophages, should incorporate fewer rNTPs in their proviral DNA, compared to HIV- 1 in macrophages. Also, we will investigate 1) preferential rNTP incorporation ?hot spots? in the HIV-1 proviral DNA sequences by using a recently developed next generation sequencing based assay, and 2) the effect of rNTP incorporation on viral mutagenesis. Finally, we will test the effect of N6-methyl adenosines (m6As) embedded in RNA templates, which affect HIV-1 mRNA expression and replication, on HIV-1 RT polymerase kinetics. This proposed work will elucidate the unique mechanistic features of HIV-1 replication in non-dividing myeloid reservoir cells, which can lead to the discovery of novel therapeutic concepts against viral persistence.